Viral hepatitis is associated with increased risk of decompensated cirrhosis or liver failure in patients positive for liver cytosol antibody type 1

Liver cytosol antibody type 1 (anti‐LC1) is reported to be a marker of type 2 autoimmune hepatitis (AIH), a type of autoimmune liver disease (AILD). However, anti‐LC1 is not entirely disease‐specific, and its clinical value in other hepatic diseases has not been well elucidated. Our study aimed to explore the associations between the diagnoses and outcome of decompensated cirrhosis or liver failure (DC/LF) in patients positive for anti‐LC1. A total of 157 patients positive for anti‐LC1 were included in our final analysis. DC/LF was defined as the outcome of patients positive for anti‐LC1. The risk of DC/LF according to diagnosis was estimated using multivariable Cox proportional hazards models, while stratified Cox regression models were used in the subgroup analyses. The diagnoses of patients positive for anti‐LC1 were found to be comprised of various liver disorders. Versus other diagnoses, viral hepatitis was associated with a 2.25‐fold increased risk of DC/LF in these patients, independent of sex, age, disease course, treatment and drinking history. Additionally, the associations were more significant by subgroup analysis in male patients, younger patients, non‐newly diagnosed patients, patients without treatment and patients without drinking history. Anti‐LC1 is not a disease‐specific antibody, as it was found in multiple types of hepatic disease. Furthermore, viral hepatitis rather than AILD was associated with an increased risk of DC/LF in patients positive for anti‐LC1. These findings emphasize the important role of viral hepatitis in the progression of DC/LF in patients positive for anti‐LC1.

Autoantibodies are essential and effective in the diagnosis and management of low-prevalence AILDs, particularly in PBC and AIH. 1,2 Liver cytosol-specific antibody type 1 (anti-LC1), against a liver-specific cytosolic component, was first detected in a proportion of patients suffering from type 2 AIH. 3  Versus other diagnoses, viral hepatitis was associated with a 2.25-fold increased risk of DC/LF in these patients, independent of sex, age, disease course, treatment and drinking history. Additionally, the associations were more significant by subgroup analysis in male patients, younger patients, non-newly diagnosed patients, patients without treatment and patients without drinking history. Anti-LC1 is not a disease-specific antibody, as it was found in multiple types of hepatic disease. Furthermore, viral hepatitis rather than AILD was associated with an increased is formiminotransferase cyclodeaminase, which catalyses the conversion of histidine to glutamic acid in liver. 2,4 AIH is a chronic inflammatory disease that, when untreated, results in cirrhosis and liver failure. The disease is comprised of at least two types according to the autoantibody profile. 5 Type 1 AIH is characterized by anti-nuclear antibody, anti-smooth muscle antibody, or autoantibodies against soluble liver antigen and liver-pancreas antigen. Contrastingly, type 2 AIH is associated with anti-liver kidney microsomal type 1 (anti-LKM1) antibody and/or anti-LC1. 6 Although this distinction between the two types was initially based on serologic antibodies, other clinical differences exist between type 1 AIH and type 2 AIH. For example, type 2 AIH mainly affects children and adolescents, and it is considerably more rare and aggressive than type 1 AIH. Additionally, females account for approximately 95% in type 2 AIH, which is substantially higher than the corresponding 75% female proportion in type 1 AIH. 7 Anti-LC1 is generally present in conjunction with anti-LKM1 in type 2 AIH, but in some cases, it may be the sole autoantibody. A previous study described 18 cases of children with AIH who exhibited serum anti-LC1 as the only autoimmune marker, and this result suggests that the presence of anti-LC1 in children with liver disease of unknown origin is strongly supportive of a diagnosis of AIH. 8 The frequency of anti-LC1 has been reported to be low. A study on an Italian cohort of AIH patients showed that the positive rate of anti-LC1 (line-blot alone) was 14.2% in adults and 35.9% in paediatric cases. 9 Another Chinese study reported a lower frequency, in which 38 patients positive for anti-LC1 were observed from 23 832 autoantibody tests (0.16%), 10 suggesting that it is difficult to investigate anti-LC1, a low-prevalence antibody, in a large population.
The disease-specificity of anti-LC1 in AIH is considered apparent, since it has not been observed in a large number of patients with other hepatic or extra-hepatic diseases. 11,12 Indeed, anti-LC1 can be detected in HCV infection. A previous study showed that 33% (6/18) of anti-LKM (pos) /HCV (pos) patients tested positive for anti-LC1 compared to 0.83% (1/120) of anti-LKM (neg) /HCV (pos) patients and 0% (0/120) of anti-LKM1 (neg) /HBV (pos) patients, suggesting that anti-LC1 screening may only have application potential for anti-LKM1 (pos) /HCV (pos) patients, given its low prevalence. 13 Additionally, anti-LC1 positivity has been reported in a small proportion of paediatric type 1 AIH and autoimmune sclerosing cholangitis patients, 2 as well as blood donors. 14 Hence, anti-LC1 is not entirely disease-specific, and its presence should be interpreted in a clinical context.
The natural history of cirrhosis is characterized by an asymptomatic compensated phase followed by a decompensated phase, which is marked by the development of overt clinical signs, the most frequent of which are bleeding, ascites, encephalopathy, and jaundice. The disease usually progresses towards death or liver transplantation. 15,16 Meanwhile, liver failure encompasses a group of clinical syndromes characterized by severe liver damage, e.g., severe decompensation of synthesis, detoxification, metabolism, and biological transformation, which is a significant clinical problem with high morbidity and mortality. 17 Hence, decompensated cirrhosis or liver failure is commonly considered an outcome of hepatic diseases.
Despite many researches on the presence of anti-LC1 in type 2 AIH, the clinical value of anti-LC1 in other types of hepatic disease, especially in the Chinese population, is not yet well understood. This retrospective study aimed to explore the associations between diagnoses and outcome of decompensated cirrhosis or liver failure (DC/LF) in patients positive for anti-LC1.

| Study design and patients
This retrospective study was conducted at Henan Provincial People's Hospital (Zhengzhou, China). A total of 214 cases positive for anti-LC1 were screened from 8838 patients from January 2017 to June 2022. These 8838 patients either had liver disease or complained of liver discomfort, thus having subsequently undergone screening for the anti-LC1 antibody. The termination of the followup was on December 31, 2022. The clinical and laboratory data of these patients were retrospectively analysed.
The exclusion criteria were described as follows: patients who were younger than 18 years of age (n = 4), patients with malignant tumours (n = 36), patients with extra-hepatic diseases (n = 10), and patients comorbid with hepatic disease and syphilis (n = 3). We excluded an additional 4 cases due to missing data. A total of 157 patients were included in the final analysis ( Figure 1).
The presence of decompensated cirrhosis or liver failure (DC/LF) was defined as the outcome of patients positive for anti-LC1, given that these patients commonly have a poor prognosis. 18,19 Patients were diagnosed according to Chinese guidelines on the management of liver cirrhosis 20 and liver failure. 21 Notably, DC/LF patients were diagnosed at the time of anti-LC1 evaluation, and the patient states did not change in the subsequent follow-up.
The diagnoses in this study were classified into 4 groups according to their clinical value and correlation with LC-1. The types of viral hepatitis included hepatitis B, hepatitis C and hepatitis E in this study (HBV: n = 45, HCV: n = 7, HEV: n = 4, HBV + HCV: n = 2, HBV + HEV: n = 2). The types of autoimmune liver disease in this study included PBC, AIH and their overlap syndrome (PBC: n = 13, AIH: n = 12, PBC + AIH: n = 19). The diagnoses of PBC and AIH were conducted in accordance with corresponding guidelines. [22][23][24] Other diagnoses included non-alcoholic fatty liver disease, drug hepatitis, hepatolenticular degeneration and unknown liver damage, etc.
In this study, disease course was classified into 4 groups (0, ≤12, 12-60 or >60 months), whereby "0" refers to cases who were newly diagnosed. Furthermore, the treatments in this study included nucleoside analogs, ribavirin ± interferon, glucocorticoid, ursodeoxycholic acid (UDCA), the combination of UDCA and glucocorticoid, and no treatment.
Drinking history was defined according to the guidelines of prevention and treatment for alcoholic liver disease. 25 One category consisted of those with a long-term history of drinking (generally more than 5 years, equivalent to ethanol ≥40 g/d in men and ≥ 20 g/d in women), while the other consisted of those with a history of heavy drinking within the 2 weeks prior (equivalent to ethanol ≥80 g/d).
The electronic medical record system was used to obtain patient information on age, sex, diagnosis, disease course, treatment, and drinking history. This study was performed according to the guidance of the Ethics Committee of Henan Provincial People's Hospital (201950).

| Laboratory indicator detection
A multiparametric line immunoassay was used for anti-LC1 detection following the manufacturer's protocols (Oumeng, Germany), and the EUROLineMaster automatic immunoblotting instrument (Oumeng, Germany) was used in this study. Grey values were automatically identified by the analyser (negative: ≤10, weakly positive: 11-25, strongly positive: ≥26).
(PLTs) were tested in an XN-10 automated haematology analyser (Sysmex). The prothrombin time was measured using the coagulation method in a CS-5100 automated coagulation analyser (Sysmex), and INR was calculated according to the manufacturer's protocols.

| Statistical analysis
Continuous variables are presented as means ± standard deviations (SD) or medians (interquartile range 25-75% [IQR]) according to the normality of the distribution for continuous variables, whereas categorical variables were presented as frequencies (%). The unpaired Student's t-test or Mann-Whitney U-test was used to analyse the differences between two groups (DC/LF and non-DC/LF) in demographic and clinical features analysis, according to the normality of the distribution for continuous variables. The chi-square test was used for categorical variables. 26 The associations of the patient outcome (DC/LF) with the variables were calculated by Cox proportional hazards models and presented as hazard ratios (HRs) and 95% confidence intervals (CIs). Additionally, the associations between diagnoses and DC/LF were evaluated by non-adjusted and multivariate-adjusted models. The crude model was the non-adjusted model with no covariates adjusted. Adjusted model I adjusted for sex and age. Adjusted model II adjusted for sex, age, disease course, treatment and drinking history. Furthermore, stratified Cox regression models were used for subgroup analyses. Meanwhile, we converted age, a continuous variable, into a categorical variable according to the clinical criteria for the elderly (60 ± years), and then performed an interaction test. Disease course was classified into newly diagnosed patients (for whom the disease course = 0) and non-newly diagnosed patients (for whom the disease course >0). The treatment subgroups included non-treatment group and treatment group. The likelihood ratio test was used to evaluate the interaction across subgroups. The abovementioned statistical methods were similar to those used in our previous report. 26 The statistical software packages R (V.3.4.3) and SPSS (V.17.0) were used in this study. P < .05 was considered statistically significant.

| Demographic and clinical features in patients positive for anti-LC1
The final analysis included 157 adult patients who followed the exclusion criteria ( Figure 1). First, the demographic, clinical, and laboratory characteristics of patients exhibiting different degrees of positivity for anti-LC1 based on its concentration were analysed (Table S1). However, no significant difference was observed between the weakly positive group and the strongly positive group regarding these different variables, suggesting there was no significant difference in the effect of different concentrations of positive anti-LC1 on the variables. Hence, we did not distinguish the different concentrations of positive anti-LC1 in subsequent analyses.
The demographic and clinical features of patients positive for anti-LC1 were demonstrated in Table 1. Notably, the outcome of 76 cases was DC/LF, accounting for 48.41% of the enrolled 157 patients. Among the patients, 47.77% (75/157) were female and the mean age of all patients was 51.7 ± 13.9 years. Additionally, the ages of DC/LF patients were significantly higher than those of non-DC/LF patients. Notably, the diagnoses of patients positive for anti-LC1 included various liver disorders, suggesting that anti-LC1 was not a disease-specific antibody, as it could be found in other types of hepatic disease. Because AILD in this study mainly included PBC, AIH, and their overlapping syndrome, here, we classified them into AILD. A significant difference was observed in the rates of DC/LF among patients with different diagnoses. Furthermore, there were significant differences between DC/LF patients and non-DC/LF patients regarding different treatments. However, no significant differences were observed between DC/LF group and non-DC/LF group regarding disease course or drinking history. These results suggest that age, diagnosis, and treatment are probably associated with the occurrence of DC/LF in patients positive for anti-LC1.

| Univariate Cox regression models in patients positive for anti-LC1
The HRs and 95% CIs for the risk of DC/LF by different variables were demonstrated in Table 2. The results showed that age was associated with a 3% increased risk of DC/LF per year (95% CIs, 1.01-1.06). Additionally, patients diagnosed with viral hepatitis, were associated with 2.13 fold increased risk of DC/LF, referenced to those with other diagnoses. Furthermore, patients treated with nucleoside analogs had a 2.8 fold increased risk of DC/LF compared to patients without treatment.

| Associations of diagnoses with the risk of DC/LF in patients positive for anti-LC1
The HRs and 95% CIs for the risk of DC/LF by diagnosis were shown in Table 3. In the non-adjusted model, an increased risk of DC/LF was observed in patients with viral hepatitis, compared to those with other diagnoses (P = .004). After adjustment for age and sex, the HR was 3.3 (95% CIs, 1.48-7.38) (P = .004). Furthermore, the HRs were 3.25 (95% CIs, 1.3-8.11) after adjustment for age, sex, disease course, treatment and drinking history (P = .012), suggesting that viral hepatitis plays an important role in the progression of DC/LF in patients positive for anti-LC1 antibody.

| Subgroup analyses
We conducted stratified and interactive analyses to investigate whether the associations between diagnoses and DC/LF were stable in different subgroups (Table 4).
We converted age, a continuous variable, into a categorical variable according to the clinical criteria for the elderly (60 ± years). Additionally, the disease course was classified into newly diagnosed patients (for whom the disease course = 0) and non-newly diagnosed patients (for whom the disease course >0). The treatment subgroups included non-treatment group and treatment group.
The data showed that the associations between diagnoses and DC/LF in the stratified analysis were consistent with that in the multivariable logistic regression analysis. The stratified analysis demonstrated a statistically significant association between viral hepatitis and DC/LF in male patients (adjusted OR, 3.88; 95% CI, 1.2-12.57), patients younger than 60 years (adjusted OR, 3.09; 95% CI, 1.19-8.04), non-newly diagnosed patients (adjusted OR, T A B L E 1 Demographic and clinical characteristics in patients positive for anti-LC1.

Variables
Total patients (n = 157)  . The other diagnosis included biliary tract diseases, non-alcoholic fatty liver disease, drug hepatitis, hepatolenticular degeneration and unknown liver damage, etc. P < .05 was considered statistically significant.
The interaction analysis revealed no interactive role in the associations between diagnoses and DC/LF in patients positive for anti-LC1.

| DISCUSSION
In this retrospective study, we explored the associations between diagnoses and outcome of DC/LF in patients positive for anti-LC1. Interestingly, anti-LC1 was proved to be not a disease-specific antibody and it could be found in several types of hepatic disease. Furthermore, viral hepatitis rather than AILD was associated with increased risk of DC/LF in patients positive for anti-LC1, independent of sex, age, disease course, treatment and drinking history. These findings suggested that viral hepatitis played an important role in the progression of DC/LF in patients positive for anti-LC1. The difference in the association of AILD or viral hepatitis with DC/LF, may be due to the characteristics of the two diseases. AILD is a generally progressive, chronic hepatitis, characterized by a fluctuating course, and appropriate management can delay disease progression, improve the quality of life, and avoid negative outcomes. 5 In contrast, viral hepatitis is caused by hepatotropic viruses that can establish a persistent and chronic infection in humans through immune anergy. 27 As a result of the ongoing necrosis and inflammation in the liver, chronic HBV infection develops into cirrhosis in up to 40% of untreated patients. 28 Most patients with HCV infection develop liver fibrosis, and 15-25% of them progress to cirrhosis after 10-40 years. 29 Anti-LC1 has mostly been reported in children and juveniles, and it is mainly associated with anti-LKM-1 reactivity. 9,12,30 However, our research focused on adult patients with liver disorders by excluding juveniles. Considering the scarcity of studies on anti-LC1 in the Asian population, our study investigated a Chinese population to reveal low prevalence of anti-LC1 in Chinese patients (2.42%, 214/8838), which was higher than the reported study (0.16%, 38/23 832), 10 and the difference in the results may be explained by population differences. Although the cooccurrence of anti-LC1 and anti-LKM1 was observed in the enrolled patients in this study, the reported lower frequency of anti-LKM1 (0.01%, 15/1 17 452) 31 compared to anti-LC1 suggested that it is more difficult to explore the association between anti-LKM1 and anti-LC1 in Chinese population.
The association between anti-LC1 and viral hepatitis is poorly understood, especially in hepatitis B. A study on a Greek cohort showed that anti-LC1 was not detected in a large cohort of anti-LKM1 negative patients with chronic hepatitis B and C infections. 13 In this study, hepatitis B, hepatitis C and hepatitis E were included in the umbrella diagnosis of viral hepatitis (HBV: n = 45, HCV: n = 7, HEV: n = 4, HBV + HCV: n = 2, HBV + HEV: n = 2). The unexpected finding that the hepatitis B virus accounted for the majority of the viral hepatitis cases (81.67%, 49/60), reveals that hepatitis B is closely related to the development of DC/LF in LC-1-positive patients, suggesting besides hepatitis C, there may be a close interaction between hepatitis B and AILD.
In this study, the associations between diagnoses and the DC/LF outcome by subgroup analysis were more significant in male patients, younger patients (<60 years), non-newly diagnosed patients (disease course >0 month), patients without treatment and patients without drinking history, suggesting the effects of different populations on outcome are heterogeneous, and emphasizing the importance of strict inclusion and exclusion criteria.
This study had certain limitations. First, the lowprevalence of anti-LC1 resulted in a small size of the enrolled Chinese population, even though more than 8000 cases were screened in this study, thus causing potential bias. Second, the diagnoses of the enrolled patients were classified into 4 groups, e.g. viral hepatitis, AILD, and other diagnoses. These other diagnoses included non-alcoholic fatty liver disease, drug hepatitis, hepatolenticular degeneration and unknown liver damage, among others. Due to the small number of cases in each disease, the associations of these diseases and DC/LF in patients positive for anti-LC1 were not analysed respectively, and the resulting bias was not evaluated. Third, patient data obtained upon baseline examination (weight, etc.) and lifestyle characteristics (physical activity, etc.), were not available in the study, thus causing bias in the results.
In conclusion, anti-LC1 was found not to be a diseasespecific antibody, as it could be found in several other types of hepatic disease. Furthermore, viral hepatitis rather than AILD was independently associated with an increased risk of DC/LF in patients positive for anti-LC1. These findings emphasize the important role of viral hepatitis in the progression of DC/LF in patients positive for anti-LC antibody. Further studies are needed to explore the potential underlying mechanisms.